A somatic cell cloned rhesus monkey obtained by trophoblast replacement [WGBS II]
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ABSTRACT: Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Somatic cell nuclear transfer (SCNT) has been used to clone cynomolgus monkeys (Macaca fascicularis), but the cloning of other non-human primate species remains to be achieved. On the other hand, our histological examination indicated severe calcification of the placenta of SCNT fetuses. Additionally, we found that some of the maternal-biased imprinting genes were aberrantly lost in the cloned embryos by multi-omics analysis. We thus developed a trophoblast replacement (TR) method, providing an ICSI-derived placenta for the SCNT fetus, to support the full-term development of the cloned embryos after implantation. By combining this TR method with treatment with epigenetic modification factors, we obtained a healthy somatic cell cloned rhesus monkey. Thus, TR represents a useful approach for rhesus monkey cloning.
Project description:Our knowledge of genomic imprinting in primates is lagging behand that of mice largely due to the difficulties of allelic analyses in outbred animals. To understand imprinting dynamics in primates, we profiled transcriptomes, DNA methylomes and H3K27me3 in uniparental monkey embryos. We further developed single-nucleotide polymorphisms (SNP)-free methods, TARSII and CARSII, to identify germline differentially methylated regions (DMRs) in somatic tissues. Our comprehensive analyses showed that allelic DNA methylation, but not H3K27me3, is a major mark that correlates with paternal-biasedly expressed genes (PEGs) in uniparental monkey embryos. Interestingly, primate germline DMRs are different from PEG-associated DMRs in early embryos and are enriched in placenta. Strikingly, most placenta-specific germline DMRs are lost in placenta of cloned monkey. Collectively, our study establishes SNP-free germline DMR identification methods, defines developmental imprinting dynamics in primates and demonstrates imprinting defects in cloned monkey placenta, which provides important clues for improving primate cloning.
Project description:Since the creation of Dolly, the first sheep cloned via somatic cell nuclear transfer (SCNT), in 1997, more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1-5%) is that the clones are likely derived from adult stem cells, which form an extremely small fraction in most adult tissues. Support for this hypothesis is that the cloning efficiency of full term development using embryonic stem (ES) cells as nuclear donors is 5-10 times higher than that for somatic cells as nuclear donors. Additionally, cloned pups could not be produced directly from cloned embryos derived from nuclei of differentiated B and T cells or neuronal cells. The question remains: can SCNT-derived animal clones be derived from truly differentiated somatic cells? We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells (HSCs), progenitor cells (HPCs), and granulocytes. Surprisingly, we found that cloning efficiency increases over the differentiation hierarchy. The terminally differentiated post-mitotic granulocytes yielded the greatest cloning efficiency and we produced two cloned pups from granulocytes. We conclude that cloned mammals could be directly derived from post-mitotic differentiated somatic cells. Keywords: cell type comparison
Project description:Animal cloning can be achieved through somatic cell nuclear transfer (SCNT), yet the success rate is very low. Recent studies have revealed H3K9me3 in donor cells and abnormal Xist activation as epigenetic barriers that impede SCNT reprogramming. Here we overcome both barriers by using Xist knockout donor cells combined with overexpressing Kdm4d and achieved the highest cloning efficiency in mice. However, post-implantation developmental defects and abnormal placenta were still observed, indicating presence of additional epigenetic barriers impedes SCNT cloning. Comparative DNA methylome analysis of IVF and SCNT blastocysts identified many abnormally methylated regions in SCNT embryos, despite successful global methylome reprogramming. Strikingly, allelic transcriptome and ChIP-seq analyses of preimplantation SCNT embryos revealed a complete loss of H3K27me3 imprinting, which likely accounts for postimplantation developmental defects of SCNT embryos. This study not only provides an efficient method for mouse cloning, but also paves the way for further improving SCNT cloning efficiency.
Project description:Animal cloning can be achieved through somatic cell nuclear transfer (SCNT), yet the success rate remains very low. Recent studies have revealed two epigenetic barriers, H3K9me3 in donor cells and abnormal Xist activation, that impede SCNT reprogramming. Here we overcome both barriers by combining the use of Xist knockout donor cells and overexpressing Kdm4d, which allowed us to achieve the highest mouse cloning efficiency. However, SCNT-associated developmental defects and abnormal placenta were still observed, suggesting the existence of additional epigenetic defects in these SCNT embryos. Comparative DNA methylome analysis of IVF and SCNT blastocysts identified many abnormally methylated regions in SCNT embryos, despite successful global methylome reprogramming. Strikingly, allelic transcriptome analyses of SCNT blastocysts revealed a complete loss-of-imprinting at the H3K27me3-dependent imprinted genes, which may account for postimplantation developmental defects of SCNT embryos. This study thus not only provides the most efficient method for mouse cloning but also points the way for further improve SCNT cloning.